Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
1.
Gene Ther ; 28(7-8): 456-468, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33612827

RESUMEN

Adeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood-brain barrier, our data suggests that, independent of their ability to cross the blood-brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.


Asunto(s)
Dependovirus , Vectores Genéticos , Animales , Sistemas CRISPR-Cas , Sistema Nervioso Central , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Ratones , Neuronas , Transducción Genética
2.
Gene Ther ; 28(10-11): 646-658, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33558692

RESUMEN

CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.


Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Animales , Sistema Nervioso Central , Edición Génica/métodos , Técnicas de Inactivación de Genes , Ratones , Neuronas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo
3.
J Immunol ; 197(10): 3806-3819, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27815440

RESUMEN

Despite being one of the most common rheumatologic diseases, there is still no disease-modifying drug for primary Sjögren's syndrome (pSS). Advancing our knowledge of the target tissue has been limited by the low dimensionality of histology techniques and the small size of human salivary gland biopsies. In this study, we took advantage of a molecularly validated mouse model of pSS to characterize tissue-infiltrating CD4+ T cells and their regulation by the lymphotoxin/LIGHT signaling axis. Novel cell subsets were identified by combining highly dimensional flow and mass cytometry with transcriptomic analyses. Pharmacologic modulation of the LTßR signaling pathway was achieved by treating mice with LTßR-Ig, a therapeutic intervention currently being tested in pSS patients (Baminercept trial NCT01552681). Using these approaches, we identified two novel CD4+ T cell subsets characterized by high levels of PD1: Prdm1+ effector regulatory T cells expressing immunoregulatory factors, such as Il10, Areg, Fgl2, and Itgb8, and Il21+ effector conventional T cells expressing a pathogenic transcriptional signature. Mirroring these observations in mice, large numbers of CD4+PD1+ T cells were detected in salivary glands from Sjögren's patients but not in normal salivary glands or kidney biopsies from lupus nephritis patients. Unexpectedly, LTßR-Ig selectively halted the recruitment of PD1- naive, but not PD1+, effector T cells to the target tissue, leaving the cells with pathogenic potential unaffected. Altogether, this study revealed new cellular players in pSS pathogenesis, their transcriptional signatures, and differential dependency on the lymphotoxin/LIGHT signaling axis that help to interpret the negative results of the Baminercept trial and will guide future therapeutic interventions.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-alfa/metabolismo , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Anfirregulina/genética , Animales , Biopsia , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Interleucina-10/genética , Interleucinas/genética , Riñón/patología , Nefritis Lúpica/inmunología , Linfotoxina-alfa/genética , Ratones , Glándulas Salivales/patología , Transducción de Señal , Síndrome de Sjögren/terapia , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores
4.
Clin Immunol ; 169: 69-79, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27352977

RESUMEN

B-cell activating factor (BAFF) levels are increased in rheumatoid arthritis, lupus and primary Sjögren's syndrome (pSS). However, BAFF contribution to pathogenesis is not completely understood. In pSS, immune infiltration of the salivary and lacrimal glands leads to xerostomia and xerophtalmia. Glandular B cell hyperactivation, differentiation into germinal center (GC)-like structures and plasma cell accumulation are histopathological hallmarks that were attributed to increased BAFF. Here, we experimentally tested this hypothesis by overexpressing BAFF in a mouse model of pSS. BAFF overexpression enhanced lymphocytic infiltration and MHCII expression on B cells. Increased BAFF also induced B cell differentiation into GC B cells within the autoimmune target tissue. However, even in these conditions, GC B cells only accounted for <1% of glandular B cells, demonstrating that BAFF is not efficiently promoting ectopic GC formation in pSS and warranting further investigation of therapeutics targeting both BAFF and the related TNF-family member APRIL.


Asunto(s)
Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Síndrome de Sjögren/inmunología , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Diferenciación Celular/genética , Células Cultivadas , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunohistoquímica , Aparato Lagrimal/inmunología , Aparato Lagrimal/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Análisis de Secuencia por Matrices de Oligonucleótidos , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Xeroftalmia/genética , Xeroftalmia/inmunología , Xeroftalmia/metabolismo , Xerostomía/genética , Xerostomía/inmunología , Xerostomía/metabolismo
5.
Cell Rep ; 43(1): 113636, 2024 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-38183652

RESUMEN

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.


Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/metabolismo , Proteoma/metabolismo , Proteómica
6.
Mol Ther Nucleic Acids ; 34: 102057, 2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-37928442

RESUMEN

Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA (pri-miRNA) scaffolds impact the potency and safety profile of the expressed amiR in vivo. In our process to develop an AAV amiR targeting SOD1, we performed a preclinical characterization of two pri-miRNA scaffolds, miR155 and miR30a, sharing the same guide strand sequence. We report that, while the miR155-based vector, compared with the miR30a-based vector, leads to a higher level of the amiR and more robust suppression of SOD1 in vitro and in vivo, it also presents significantly greater risks for CNS-related toxicities in vivo. Despite miR30a-based vector showing relatively lower potency, it can significantly delay the development of ALS-like phenotypes in SOD1-G93A mice and increase survival in a dose-dependent manner. These data highlight the importance of scaffold selection in the pursuit of highly efficacious and safe amiRs for RNA interference gene therapy.

7.
FASEB J ; 25(5): 1664-79, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21321189

RESUMEN

Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of human type 2 diabetes (T2DM). Although SIRT1 has a therapeutic effect on metabolic deterioration in T2DM, the precise mechanisms by which SIRT1 improves insulin resistance remain unclear. Here, we demonstrate that adenovirus-mediated overexpression of SIRT1 in the liver of diet-induced insulin-resistant low-density lipoprotein receptor-deficient mice and of genetically obese ob/ob mice attenuates hepatic steatosis and ameliorates systemic insulin resistance. These beneficial effects were associated with decreased mammalian target of rapamycin complex 1 (mTORC1) activity, inhibited the unfolded protein response (UPR), and enhanced insulin receptor signaling in the liver, leading to decreased hepatic gluconeogenesis and improved glucose tolerance. The tunicamycin-induced splicing of X-box binding protein-1 and expression of GRP78 and CHOP were reduced by resveratrol in cultured cells in a SIRT1-dependent manner. Conversely, SIRT1-deficient mouse embryonic fibroblasts challenged with tunicamycin exhibited markedly increased mTORC1 activity and impaired ER homeostasi and insulin signaling. These effects were abolished by mTORC1 inhibition by rapamycin in human HepG2 cells. These studies indicate that SIRT1 serves as a negative regulator of UPR signaling in T2DM and that SIRT1 attenuates hepatic steatosis, ameliorates insulin resistance, and restores glucose homeostasis, largely through the inhibition of mTORC1 and ER stress.


Asunto(s)
Retículo Endoplásmico/metabolismo , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Sirtuina 1/metabolismo , Animales , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Prueba de Tolerancia a la Glucosa , Células Hep G2 , Humanos , Immunoblotting , Inmunohistoquímica , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Obesidad , Receptores de LDL/genética , Receptores de LDL/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genética , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología
8.
Biochem Biophys Res Commun ; 410(3): 543-8, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21683058

RESUMEN

Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1ß-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1ß-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1ß, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE(-/-)) mice. VCAM-1 and iNOS expression were higher in ApoE(-/-) than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE(-/-) mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-κB crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.


Asunto(s)
Angiotensina II/fisiología , Enfermedades Cardiovasculares/genética , Regulación de la Expresión Génica , Inflamación/genética , Músculo Liso Vascular/metabolismo , Angiotensina II/farmacología , Animales , Aorta , Apolipoproteínas E/genética , Enfermedades Cardiovasculares/metabolismo , Regulación hacia Abajo , Fosfatasa 1 de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Inflamación/metabolismo , Interleucina-1beta/farmacología , Interleucina-1beta/fisiología , Ratones , Ratones Mutantes , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Molécula 1 de Adhesión Celular Vascular
9.
J Cardiovasc Pharmacol ; 58(3): 263-71, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21654327

RESUMEN

Our purpose was to determine if high-fat diet and treatment with a polyphenol regulate the acetylation of lysine-382 of p53, the site regulated by sirtuin-1, and apoptosis in the endothelium of the atherosclerotic lesion-prone mouse aortic arch. In cultured endothelial cells, 2 atherogenic stimuli, hydrogen peroxide and tumor necrosis factor-α, increased the acetylation of p53 lysine-382, and caspase-3 cleavage, an indicator of apoptotic signaling. The polyphenol, S17834, significantly prevented these changes. In low-density lipoprotein receptor-deficient mice, a high-fat diet increased, and treatment with S17834 attenuated early atherosclerotic lesions on the lesser curvature of the aortic arch. In wild-type C57BL6 mice fed the same diet, no atherosclerotic lesions were observed in this lesion-prone area, but p53 acetylation and caspase-3 cleavage increased in the endothelium. In high-fat fed mice, S17834 increased sirtuin-1 protein in the lesion-prone endothelium and prevented both the increase in p53 acetylation and caspase-3 cleavage without affecting blood lipids. These results indicate that high-fat diet increases and S17834 decreases the acetylation of p53 in lesion-prone aortic endothelial cells of normal mice independently of blood lipids, suggesting that the polyphenol may regulate endothelial cell p53 acetylation and apoptosis via local actions.


Asunto(s)
Aterosclerosis/tratamiento farmacológico , Benzopiranos/farmacología , Dieta Alta en Grasa , Hipolipemiantes/farmacología , Polifenoles/farmacología , Sirtuina 1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Apoptosis , Aterosclerosis/enzimología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Benzopiranos/metabolismo , Benzopiranos/farmacocinética , Caspasa 3/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Hipolipemiantes/metabolismo , Lípidos/sangre , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polifenoles/metabolismo , Polifenoles/farmacocinética , Transducción de Señal , Superóxidos/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/química
10.
Arterioscler Thromb Vasc Biol ; 28(1): 127-34, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18032781

RESUMEN

OBJECTIVE: Activation of thromboxane receptors (TPr) is implicated in atherosclerosis and inflammation. This study examined how activation of TPr modulates IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression in aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: In VSMCs, activation of TPr with U46619, a stable thromboxane A2 mimetic, alone did not induce VCAM-1 expression, but enhanced that caused by IL-1beta. The enhancement of VCAM-1 expression caused by U46619 occurred at the transcriptional level and was inhibited either by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, or by overexpression of a dominant-negative JNK1, but not by SB203580, a p38 mitogen-activated protein kinase inhibitor. The activation of JNK by U46619 resulted in enhanced phosphorylation and nuclear translocation of c-Jun associated with an enhanced activation of activator protein (AP)-1, which were abolished by SQ29548, a TPr antagonist, or the JNK inhibitor. Treatment of the cells with U46619 alone did not induce NF-kappaB activation. Furthermore, U46619 enhanced IL-1beta-induced THP-1 monocyte binding to VSMCs, which was inhibited by SQ29548 or SP600125. CONCLUSIONS: This study demonstrates that activation of TPr upregulates IL-1beta-induced VCAM-1 expression by enhancing the activation of JNK pathway that leads to enhanced AP-1 activation.


Asunto(s)
Aorta Torácica/citología , Interleucina-1beta/fisiología , Células Musculares/fisiología , Receptores de Tromboxanos/fisiología , Transducción de Señal/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Células Cultivadas , Eicosanoides/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Músculo Liso Vascular/citología , Ratas , Ratas Wistar , Factor de Transcripción AP-1/fisiología , Regulación hacia Arriba
11.
Free Radic Biol Med ; 45(6): 756-62, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18590812

RESUMEN

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.


Asunto(s)
Aorta/metabolismo , Cisteína/metabolismo , Diabetes Mellitus Experimental/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Diabetes Mellitus Experimental/enzimología , Electroforesis en Gel de Poliacrilamida , Masculino , Datos de Secuencia Molecular , Oxidación-Reducción , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Espectrometría de Masa por Ionización de Electrospray , Porcinos , Espectrometría de Masas en Tándem
12.
Diabetes ; 55(8): 2180-91, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16873680

RESUMEN

Because polyphenols may have beneficial effects on dyslipidemia, which accelerates atherosclerosis in diabetes, we examined the effect of polyphenols on hepatocellular AMP-activated protein kinase (AMPK) activity and lipid levels, as well as hyperlipidemia and atherogenesis in type 1 diabetic LDL receptor-deficient mice (DMLDLR(-/-)). In HepG2 hepatocytes, polyphenols, including resveratrol (a major polyphenol in red wine), apigenin, and S17834 (a synthetic polyphenol), increased phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC), and they increased activity of AMPK with 200 times the potency of metformin. The polyphenols also prevented the lipid accumulation that occurred in HepG2 cells exposed to high glucose, and their ability to do so was mimicked and abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Furthermore, treatment of DMLDLR(-/-) mice with S17834 prevented the decrease in AMPK and ACC phosphorylation and the lipid accumulation in the liver, and it also inhibited hyperlipidemia and the acceleration of aortic lesion development. These studies 1) reveal that inactivation of hepatic AMPK is a key event in the pathogenesis of hyperlipidemia in diabetes, 2) point to a novel mechanism of action of polyphenols to lower lipids by activating AMPK, and 3) emphasize a new therapeutic avenue to benefit hyperlipidemia and atherosclerosis specifically in diabetes via activating AMPK.


Asunto(s)
Aterosclerosis/prevención & control , Diabetes Mellitus Experimental/complicaciones , Flavonoides/administración & dosificación , Lípidos/sangre , Complejos Multienzimáticos/metabolismo , Fenoles/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de LDL/deficiencia , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Animales , Apigenina/farmacología , Benzopiranos/administración & dosificación , Carcinoma Hepatocelular , Línea Celular Tumoral , Diabetes Mellitus Experimental/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Glucosa/farmacología , Humanos , Hipolipemiantes/administración & dosificación , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Polifenoles , Receptores de LDL/fisiología , Resveratrol , Estilbenos/administración & dosificación
13.
Diabetes ; 55(1): 110-9, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16380483

RESUMEN

Arachidonic acid metabolites, some of which may activate thromboxane A(2) receptors (TPr) and contribute to the development of diabetes complications, including nephropathy, are elevated in diabetes. This study determined the effect of blocking TPr with S18886 or inhibiting cyclooxygenase with aspirin on oxidative stress and the early stages of nephropathy in streptozotocin-induced diabetic apolipoprotein E(-/-) mice. Diabetic mice were treated with S18886 (5 mg . kg(-1) . day(-1)) or aspirin (30 mg . kg(-1) . day(-1)) for 6 weeks. Neither S18886 nor aspirin affected hyperglycemia or hypercholesterolemia. There was intense immunohistochemical staining for nitrotyrosine in diabetic mouse kidney. In addition, a decrease in manganese superoxide dismutase (MnSOD) activity was associated with an increase in MnSOD tyrosine-34 nitration. Tyrosine nitration was significantly reduced by S18886 but not by aspirin. Staining for the NADPH oxidase subunit p47(phox), inducible nitric oxide synthase, and 12-lipoxygenase was increased in diabetic mouse kidney, as were urine levels of 12-hydroxyeicosatetraenoic acid and 8-iso-prostaglandin F(2alpha). S18886 attenuated all of these markers of oxidant stress and inflammation. Furthermore, S18886 significantly attenuated microalbuminuria in diabetic mice and ameliorated histological evidence of diabetic nephropathy, including transforming growth factor-beta and extracellular matrix expression. Thus, in contrast to inhibiting cyclooxygenase, blockade of TPr may have therapeutic potential in diabetic nephropathy, in part by attenuating oxidative stress.


Asunto(s)
Apolipoproteínas E/deficiencia , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Naftalenos/farmacología , Estrés Oxidativo/efectos de los fármacos , Propionatos/farmacología , Proteinuria/metabolismo , Receptores de Tromboxanos/antagonistas & inhibidores , Animales , Apolipoproteínas E/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Aspirina/farmacología , Femenino , Eliminación de Gen , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Ratones , NADPH Oxidasas , Naftalenos/uso terapéutico , Óxido Nítrico Sintasa de Tipo II , Fosfoproteínas/metabolismo , Propionatos/uso terapéutico , Proteinuria/complicaciones , Proteinuria/tratamiento farmacológico , Receptores de Tromboxanos/metabolismo
14.
Arterioscler Thromb Vasc Biol ; 26(4): 910-6, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16439706

RESUMEN

OBJECTIVE: Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that is a major source of cellular NADPH. The purpose of this study was to examine whether G6PD deficiency affects vascular oxidants and atherosclerosis in high-fat fed apolipoprotein (apo) E(-/-) mice. METHODS AND RESULTS: G6PD-mutant mice whose G6PD activity was 20% of normal were crossbred with apoE(-/-) mice. Among male apoE(-/-) mice that were fed a western-type diet for 11 weeks, G6PD wild-type (E-WT), and G6PD hemizygous (E-Hemi) mice were compared. Basal blood pressure was significantly higher in E-Hemi. However, superoxide anion release, nitrotyrosine, vascular cell adhesion molecule (VCAM)-1, and inducible nitric oxide synthase immunohistochemical staining were less in E-Hemi compared with E-WT aorta. Serum cholesterol level was lower in E-Hemi, but aortic lesion area was decreased in E-Hemi even after adjusting for serum cholesterol. CONCLUSIONS: Lower NADPH production in G6PD deficiency may result in lower NADPH oxidase-derived superoxide anion, and thus lower aortic lesion growth. The association of higher blood pressure with lower serum cholesterol levels in this mouse model is indicative of the complex effects that G6PD deficiency may have on vascular disease.


Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Aterosclerosis/patología , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/patología , Superóxidos/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/complicaciones , Dieta Aterogénica , Regulación hacia Abajo , Deficiencia de Glucosafosfato Deshidrogenasa/complicaciones , Masculino , Ratones , Ratones Noqueados , Mutación , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo
16.
Circulation ; 112(2): 257-63, 2005 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-15998684

RESUMEN

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) regulates production of the reduced form of NADPH through the pentose phosphate pathway. G6PD may therefore affect superoxide anion production via vascular NADPH oxidase, which is key in mediating the vascular response to angiotensin II (Ang II). We determined the hypertensive and vascular hypertrophic response to Ang II in G6PD-deficient mice. METHODS AND RESULTS: Ang II (0.7 mg/kg per day) was infused via subcutaneous osmotic pumps for 6 days in male hemizygote G6PD mutant (G6PD(mut)) and wild-type (WT) C3H mice. (1) Compared with WT, G6PD(mut) mouse aorta had 10% to 20% of G6PD activity and 50% less NADPH. (2) Basal systolic blood pressure was not significantly different in G6PD(mut) mice (WT 88+/-4 mm Hg versus G6PD(mut) 95+/-4 mm Hg), but Ang II increased blood pressure to a lower level in G6PD(mut) mice (WT 139+/-4 mm Hg versus G6PD(mut) 123+/-5 mm Hg; P<0.05). (3) Ang II increased aortic medial thickness less in G6PD(mut) mice (WT 71+/-2 mum versus G6PD(mut) 62+/-1 mum; P<0.01). (4) 3-o-Nitrotyrosine staining and dihydroethidium oxidation in the aorta was increased by Ang II less in G6PD(mut) mice. (5) Smooth muscle cells isolated from G6PD(mut) mice showed less Ang II-induced phosphorylation of Akt and p42/44 ERK. CONCLUSIONS: G6PD deficiency may reduce vascular superoxide anion production by limiting production of the substrate for NADPH oxidase, thereby inhibiting oxidant-mediated Ang II-induced signaling pathways that contribute to hypertension and smooth muscle hypertrophy.


Asunto(s)
Angiotensina II/farmacología , Vasos Sanguíneos/efectos de los fármacos , Deficiencia de Glucosafosfato Deshidrogenasa/fisiopatología , Angiotensina II/administración & dosificación , Animales , Aorta/patología , Vasos Sanguíneos/fisiopatología , Hipertensión/etiología , Hipertrofia/etiología , Masculino , Ratones , Ratones Mutantes , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Superóxidos/metabolismo
17.
Circulation ; 112(19): 3001-8, 2005 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-16260636

RESUMEN

BACKGROUND: S18886 is an orally active thromboxane A2 (TXA2) receptor (TP) antagonist in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. We previously showed that S18886 inhibits atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice by a mechanism independent of platelet-derived TXA2. Atherosclerosis is accelerated by diabetes and is associated with increased TXA(2) and other eicosanoids that stimulate TP. The purpose of this study was to determine whether S18886 lessens the enhanced atherogenesis in diabetic apoE(-/-) mice. METHODS AND RESULTS: Diabetes mellitus was induced in apoE(-/-) mice with streptozotocin and was treated or not with S18886 (5 mg.kg(-1).d(-1)). After 6 weeks, aortic lesion area was increased >4-fold by diabetes in apoE(-/-) mice, associated with similar increases in serum glucose and cholesterol. S18886 largely prevented the diabetes-related increase in lesion area without affecting the hyperglycemia or hypercholesterolemia. S18886 prevented deterioration of endothelial function and endothelial nitric oxide synthase expression, as well as increases in intimal markers of inflammation associated with diabetes. In human aortic endothelial cells in culture, S18886 also prevented the induction of vascular cell adhesion molecule-1 and prevented the decrease in endothelial nitric oxide synthase expression caused by high glucose. CONCLUSIONS: The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.


Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Diabetes Mellitus Experimental/complicaciones , Angiopatías Diabéticas/prevención & control , Naftalenos/farmacología , Propionatos/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Animales , Cruzamientos Genéticos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
18.
Circ Res ; 90(10): 1114-21, 2002 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-12039802

RESUMEN

Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca(2+) uptake via sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function. To study the effect of antioxidants, which are known to improve endothelium-dependent relaxation in HC, smooth muscle SERCA activity and NO-induced relaxation were studied in rabbits fed normal chow or a 0.5% cholesterol diet for 13 weeks. The antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with the HC diet in the last 3 weeks. HC impaired acetylcholine- and NO-induced relaxation, and these were restored by BHT. After inhibiting SERCA with thapsigargin, no difference existed in NO-induced relaxation among the three groups. Reduced aortic SERCA activity in HC was restored by BHT without changing SERCA protein expression. 3-Nitrotyrosine was notably increased in the media of the HC aorta, where it colocalized with SERCA. Tyrosine-nitrated SERCA protein was immunoprecipitated in the aortas of HC rabbits, where it was decreased by BHT, and it was also detected in the aortas of atherosclerotic humans. Thus, the antioxidant reverses impaired smooth muscle SERCA function in HC, and this is correlated with the improved relaxation to NO. These beneficial effects may depend on reducing the direct effects on SERCA of reactive oxygen species that are augmented in HC.


Asunto(s)
Antioxidantes/farmacología , Hidroxitolueno Butilado/farmacología , ATPasas Transportadoras de Calcio/fisiología , Hipercolesterolemia/metabolismo , Hipercolesterolemia/fisiopatología , Músculo Liso Vascular/enzimología , Tirosina/análogos & derivados , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiopatología , Calcio/metabolismo , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Humanos , Hipercolesterolemia/enzimología , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/farmacología , Estrés Oxidativo , Conejos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tirosina/metabolismo , Vasodilatación , Vasodilatadores/farmacología
19.
PLoS One ; 11(7): e0158888, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27391784

RESUMEN

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.


Asunto(s)
Antineoplásicos/farmacología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Sarcoma Sinovial/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Sarcoma Sinovial/genética , Sarcoma Sinovial/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
20.
Arterioscler Thromb Vasc Biol ; 22(11): 1811-6, 2002 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-12426209

RESUMEN

OBJECTIVE: Activation of extracellular signal-regulated kinases (ERKs) is required for interleukin-1beta to persistently activate nuclear factor (NF)-kappaB and concomitantly express inducible NO synthase (iNOS) in rat vascular smooth muscle cells (VSMCs). The present study examined whether platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) could influence the VSMC response to interleukin-1beta via an ERK-related signaling pathway. METHODS AND RESULTS: Treatment of VSMCs with PDGF or EGF alone potently induced ERK phosphorylation and DNA synthesis but did not induce NF-kappaB activation or iNOS expression. However, either PDGF or EGF markedly enhanced interleukin-1beta-induced persistent NF-kappaB activation and iNOS expression but did not affect the early and transient NF-kappaB activation. Growth factor-induced DNA synthesis was attenuated in the presence of interleukin-1beta. Inhibition of ERK phosphorylation with selective inhibitors (PD98059 or U0126) attenuated interleukin-1beta-induced persistent NF-kappaB activation and iNOS expression in either the absence or presence of the growth factors. CONCLUSIONS: These results indicate that interleukin-1beta-induced expression of NF-kappaB-dependent genes, such as iNOS, is potentiated in the presence of growth factors through a mechanism requiring ERK-dependent enhanced NF-kappaB activation, and the results also suggest that NF-kappaB activation is not required for PDGF or EGF to trigger DNA synthesis in VSMCs.


Asunto(s)
Sustancias de Crecimiento/metabolismo , Interleucina-1/fisiología , Músculo Liso Vascular/química , FN-kappa B/metabolismo , Animales , Aorta Torácica/química , Aorta Torácica/citología , Aorta Torácica/enzimología , Células Cultivadas , ADN/biosíntesis , ADN/fisiología , Activación Enzimática/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , FN-kappa B/fisiología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ratas , Transducción de Señal/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA